Investigating the venom components of the Giant Indian Centipede for Nav1.7 channel modifiers

The electro-genesis property of voltage-gated sodium channels makes them indispensable therapeutic targets for numerous conductance disorders. The sodium channel (NaV)1.7 subtype expressed in sensory neurons in the dorsal root ganglia is thought to be a crucial signaling protein involved in nociceptive pain. Centipede envenomation results in instant rigid paralysis in prey and excruciating painful stings in humans. Compared to other venomous clades, these venoms have been largely ignored due to the minimal clinical significance of the stings. However, because many animal venoms are used to probe ion channels and some are progressing to becoming modern day venom-based drugs, we extracted the venoms of both male and female Scolopendra hardickei centipedes to evaluate their composition, to investigate activities at the NaV1.7 pain amplifying channel, and to search for the presence of pore forming toxins. The properties of the poorly characterised sodium currents inherently expressed by the TE671 cell line were initially studied using whole-cell patch-clamp to aid in NaV subtype identification. The sodium currents of the TE671 cell line were completely blocked by tetrodotoxin at a concentration of 1µM and IC50 values of 2.8 nM for Huwentoxin IV and 0.8 nM for Protoxin II were obtained. The voltage at which half the number of available channels were activated (V50.act value) was recorded as -31.89 ± 1.12 mV, which is comparable to other values reported in the literature for NaV1.7. The voltage at which half the number of available channels became inactivated (V50.inact) was recorded as -69.6 ± 1.02 mV which is similar to literature values for NaV1.7. Combined, these data indicate that the subtype expressed in TE671 cells is NaV1.7. To assess the degree of modification on NaV1.7 by S. hardwickei venom components, data from macroscopic current, channel activation and channel inactivation were analysed in the absence and presence of crude venoms, separated fractions and synthetic venom peptides. A total of 96 fractions were isolated using reversed-phase high performance liquid chromatography from the venom of each sex and pooled into cocktails of twelves. The V50.act value was shifted in the depolarising direction by pooled M1-12, in the hyperpolarising direction by M61-72 and M73-84, whereas the V50.inact value was shifted in the depolarised direction by M25-36, F1-12, and F13-24 and to the hyperpolarising direction by F49-60. Two of the four peptides identified from pooled M61-72 fractions displaying the same cysteine pattern were synthesised. Peptide I induced a concentration-dependent hyperpolarising shift in activation, shifting V50.act from -35.91 ± 1.20 mV to -43.94 ± 1.08 mV at 100 nM. On the other hand, Peptide IV caused a hyperpolarising shift in V50.inact with significant alterations resulting from concentrations as low as 1 nM. Using proteomic analyses of both male and female venoms and transcriptomic analyses of venom glands and leg muscles, we have shown that there are substantial differences in the relative abundance and expression levels of high versus low molecular weight components and that over 60% of all venom components can be unique to a single sex. A total of 37 peptide and protein families were identified with a 70 and 41% uniqueness to the male venom of peptide and protein composition respectively compared to the female. These results demonstrate that S. hardwickei contains a highly sexually dimorphic venom made of neuropeptides capable of modifying the activity of NaV1.7 channel in a manner that promotes either a gain in function through the hyperpolarising shift in activation or a loss in function through a hyperpolarising shift in activation. This suggests the presence of a range of voltage gating modifiers for NaV1.7 in the venoms of S. hardwickei and provide a basis for further investigations on the location of the peptide binding sites